Interspecific polymorphisms of the 16S rRNA gene (rDNA) are widely used for species identification of mycobacteria. 16S rDNA sequences, however, do not vary greatly within a species, and they are either indistinguishable in some species, for example, in Mycobacterium kansasii and M. gastri, or highly similar, for example, in M. malmoense and M. szulgai. We determined 16S-23S rDNA internal transcribed spacer (ITS) sequences of 60 strains in the genus Mycobacterium representing 13 species (M. avium, M. conspicuum, M. gastri, M. genavense, M. kansasii,M. malmoense, M. marinum, M. shimoidei, M. simiae, M. szulgai,M. triplex, M. ulcerans, and M. xenopi). An alignment of these sequences together with additional sequences available in the EMBL database (for M. intracellulare, M. phlei, M. smegmatis, and M. tuberculosis) was established according to primary- and secondary-structure similarities. Comparative sequence analysis applying different treeing methods grouped the strains into species-specific clusters with low sequence divergence between strains belonging to the same species (0 to 2%). The ITS-based tree topology only partially correlated to that based on 16S rDNA, but the main branching orders were preserved, notably, the division of fast-growing from slowly growing mycobacteria, separate branching for M. simiae, M. genavense, and M. triplex, and distinct branches for M. xenopi and M. shimoidei. Comparisons of M. gastri with M. kansasii and M. malmoense with M. szulgairevealed ITS sequence similarities of 93 and 88%, respectively.M. marinum and M. ulcerans possessed identical ITS sequences. Our results show that ITS sequencing represents a supplement to 16S rRNA gene sequences for the differentiation of closely related species. Slowly growing mycobacteria show a high sequence variation in the ITS; this variation has the potential to be used for the development of probes as a rapid approach to mycobacterial identification.

A novel genus-specific PCR for mycobacteria with simple identification to the species level by restriction fragment length polymorphism (RFLP) was established using the 16S-23S ribosomal RNA gene (rDNA) spacer as a target. Panspecificity of primers was demonstrated on the genus level by testing 811 bacterial strains (122 species in 37 genera from 286 reference strains and 525 clinical isolates). All mycobacterial isolates (678 strains among 48 defined species and 5 indeterminate taxons) were amplified by the new primers. Among nonmycobacterial isolates, only Gordonia terrae was amplified. The RFLP scheme devised involves estimation of variable PCR product sizes together with HaeIII and CfoI restriction analysis. It yielded 58 HaeIII patterns, of which 49 (84%) were unique on the species level. Hence,HaeIII digestion together with CfoI results was sufficient for correct identification of 39 of 54 mycobacterial taxons and one of three or four of seven RFLP genotypes found inMycobacterium intracellulare and Mycobacterium kansasii, respectively. Following a clearly laid out diagnostic algorithm, the remaining unidentified organisms fell into five clusters of closely related species (i.e., the Mycobacterium aviumcomplex or Mycobacterium chelonae-Mycobacterium abscessus) that were successfully separated using additional enzymes (TaqI, MspI, DdeI, orAvaII). Thus, next to slowly growing mycobacteria, all rapidly growing species studied, including M. abscessus,M. chelonae, Mycobacterium farcinogenes,Mycobacterium fortuitum, Mycobacterium peregrinum, and Mycobacterium senegalense (with a very high 16S rDNA sequence similarity) were correctly identified. A high intraspecies sequence stability and the good discriminative power of patterns indicate that this method is very suitable for rapid and cost-effective identification of a wide variety of mycobacterial species without the need for sequencing. Phylogenetically, spacer sequence data stand in good agreement with 16S rDNA sequencing results, as was shown by including strains with unsettled taxonomy. Since this approach recognized significant subspecific genotypes while identification of a broad spectrum of mycobacteria rested on identification of one specific RFLP pattern within a species, this method can be used by both reference (or research) and routine laboratories.

A pathogenic scotochromogenic Mycobacterium xenopi-like organism was isolated from the lung of an immunocompetent young woman. This pathogen caused severe bilateral cavitary lung disease, making two surgical interventions necessary after years of chronic disease. This case prompted us to characterize this mycobacterium by a polyphasic taxonomic approach. The isolate contained chemotaxonomic markers which were typical for the genus Mycobacterium, i.e., themeso isomer of 2,6-diaminopimelic acid, arabinose, and galactose as diagnostic whole-cell sugars, MK-9(H2) as the principal isoprenoid quinone, a mycolic acid pattern of α-mycolates, ketomycolates, and wax ester mycolates, unbranched saturated and unsaturated fatty acids plus a significant amount of tuberculostearic acid, and small amounts of a C20:0 secondary alcohol. On the basis of its unique 16S rRNA and 16S-23S spacer gene sequences, we propose that the isolate should be assigned to a new species,Mycobacterium heckeshornense. This novel species is phylogenetically closely related to M. xenopi. The type strain of M. heckeshornense is strain S369 (DSM 44428T). The GenBank accession number of the 16S rRNA gene of M. heckeshornense is AF174290.